DNA double-strand break (DSB) repair is critical to prevent persistent DNA damage, counteract genome instability and suppress tumor development. By re-joining broken DNA ends, DSB repair has also the potential to produce chromosome translocations and complex rearrangements. In this project, we will investigate the mechanism and regulation of the earliest step of DSB repair, DNA end-processing and its impact on DSB repair choice, particularly microhomology-mediated end-joining (MMEJ). We hypothesize that persistent, short ssDNA overhangs is the aberrant DNA repair intermediate involved in MMEJ, the pathogenic form of DSB repair responsible for a significant fraction of tumorigenic chromosome translocations. First, we will delineate the conditions that favor the generation of these intennediates in cell-free extracts derived from Xenopus. We will evaluate the contribution of the three resection pathways: CtlP, EX01 and DN/^2. Next, we will assess the impact of cell cycle (CDK) and DNA damage (PIKK) protein kinases on these pathways and on DNA processing. Finally, we will evaluate how DNA resection influences MMEJ in human breast tumor cells with fully sequenced genomes and documented chromosome translocation landscapes. The biochemical findings gained from this project will inform experiments in all other projects of the program. The proposed experiments should provide an unprecedented level of understanding of the mechanism and regulation of resection of ligatable DNA ends as well as damaged DNA ends or DNA ends harboring adducts, such as those generated following chemo- or radiotherapy. In turn this will guide future studies aiming at decreasing the impact of chromosome translocations and improving the efficiency of cancer therapies based on the generation of DNA DSBs. RELEVANCE (See instructions): Aberrant chromosome rearrangements, particulariy chromosome translocations, are implicated in the development of human cancers. Translocations often arise from a pathogenic form of DNA repair that generates short single-strand DNA intermediates with microhomologies (MH). The goal of this project is to determine how resection and the DNA repair protein, CtlP, participate in generating persistent, toxic repair intermediates with MH leading to chromosome translocations causing tumor development.